Recombinant Photobacterium profundum Nucleoid-associated protein PBPRA2585 (PBPRA2585)

What is the functional significance of PBPRA2585 in Photobacterium profundum?

Nucleoid-associated proteins play crucial roles in bacterial chromosome organization and gene regulation. While specific information about PBPRA2585 is limited in current literature, it likely contributes to DNA compaction, gene expression regulation, and possibly pressure adaptation mechanisms in P. profundum. Like other nucleoid-associated proteins, PBPRA2585 likely interacts with DNA in specific or non-specific patterns to influence chromosome architecture.

The functional characterization should include:

• DNA binding assays under varying pressure conditions

• Phenotypic analysis of PBPRA2585 knockout strains

• Transcriptomic profiling comparing wildtype and mutant strains

• Chromatin immunoprecipitation studies to identify genomic binding sites

Based on studies of other proteins in P. profundum, pressure adaptation involves significant protein regulation, with many proteins being up- or down-regulated in response to pressure changes . As P. profundum adapts to different marine environments with fundamental physical differences, nucleoid-associated proteins like PBPRA2585 may play important roles in this adaptation process.

What methods are recommended for reconstitution of recombinant PBPRA2585?

For optimal reconstitution of recombinant PBPRA2585, follow this methodological approach:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom

• Reconstitute the lyophilized protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 40-50% to enhance stability for long-term storage

• Prepare small working aliquots to minimize freeze-thaw cycles

For functional studies requiring active protein:

• Perform buffer exchange into a DNA-binding buffer containing 20-50 mM Tris-HCl (pH 7.5-8.0), 50-150 mM NaCl, 1-5 mM MgCl₂, and 1-10% glycerol

• Verify protein concentration using Bradford or BCA assay

• Assess protein activity immediately using DNA-binding assays

• Monitor protein stability at different temperatures and pressures relevant to experimental conditions

The integrity of the reconstituted protein should be verified by SDS-PAGE with Coomassie staining, confirming >85% purity as typically observed with recombinant proteins .

How should PBPRA2585 be stored for optimal stability?

The stability of PBPRA2585 depends on proper storage conditions, which should be carefully maintained:

• For lyophilized protein:

  • Store at -20°C or -80°C for up to 12 months

  • Keep in a desiccated environment to prevent hydration

  • Avoid temperature fluctuations during storage

• For reconstituted protein:

  • Store at -20°C or -80°C in 40-50% glycerol for up to 6 months

  • Prepare multiple small aliquots to avoid repeated freeze-thaw cycles

  • For short-term use (up to one week), working aliquots can be stored at 4°C

• Stability monitoring:

  • Periodically check protein activity through functional assays

  • Assess protein integrity by SDS-PAGE after extended storage

  • Document batch-to-batch variation in stability

The shelf life is affected by multiple factors including buffer composition, storage temperature, and the intrinsic stability of the protein itself . Each new preparation should be validated for stability under the specific experimental conditions being used.

What expression patterns would be expected for PBPRA2585 under different pressure conditions?

Based on proteomic studies of P. profundum under different pressure conditions, the following patterns might be expected for PBPRA2585:

• Differential expression patterns:

  • P. profundum proteins show significant regulation in response to pressure changes

  • Many proteins are specifically up- or down-regulated between atmospheric (0.1 MPa) and high pressure (28 MPa) conditions

  • As a nucleoid-associated protein, PBPRA2585 may show pressure-dependent expression to facilitate genomic adaptation to different environments

• Experimental approach to determine expression patterns:

  • Use label-free quantitative proteomics with LC-MS analysis

  • Apply appropriate statistical criteria: detection by two or more peptides, absolute ratio of at least 1.5, p<0.05

  • Compare results with transcriptomic data, noting that discrepancies between protein and mRNA levels have been observed in P. profundum

• Expected functional implications:

  • Changes in expression may correlate with altered DNA binding and chromosome organization

  • Expression patterns might reflect adaptation to specific depth ranges in the marine environment

  • Co-regulation with other pressure-responsive proteins could indicate functional relationships

How can researchers investigate the DNA-binding properties of PBPRA2585 under variable pressure conditions?

Investigating DNA-binding properties under pressure requires specialized methodological approaches:

• High-pressure electrophoretic mobility shift assays (HP-EMSA):

  • Modify standard EMSA protocols using pressure-resistant chambers

  • Test binding to both specific and non-specific DNA sequences

  • Quantify binding affinity (Kd) at different pressures (0.1 MPa, 10 MPa, 28 MPa)

  • Include competition assays to determine sequence specificity

• Fluorescence-based high-pressure binding assays:

  • Use fluorescently labeled DNA probes

  • Monitor changes in fluorescence anisotropy or FRET under pressure

  • Employ stopped-flow systems compatible with high-pressure cells

  • Calculate binding kinetics (kon and koff) as a function of pressure

• Structural analysis under pressure:

  • High-pressure NMR studies of PBPRA2585-DNA complexes

  • Small-angle X-ray scattering to detect pressure-induced conformational changes

  • Molecular dynamics simulations incorporating pressure effects

• Comparative analysis table:

Since P. profundum shows significant adaptations to different pressure environments , these methodologies would help elucidate how PBPRA2585 contributes to pressure-responsive gene regulation through altered DNA-binding properties.

What mass spectrometry-based approaches are optimal for quantifying PBPRA2585 expression levels under different pressure conditions?

For robust quantification of PBPRA2585 expression levels, implement these MS-based approaches:

• Sample preparation considerations:

  • Harvest cells while maintaining pressure conditions until protein extraction

  • Include phosphatase and protease inhibitors to preserve post-translational modifications

  • Employ consistent lysis methods across all pressure conditions

  • Fractionate samples to enrich for nucleoid-associated proteins

• LC-MS/MS protocol for label-free quantitation:

  • Extract multi-charged ions (2+, 3+, 4+) from each LC-MS run

  • Select the five most intense MS/MS spectra per feature

  • Apply a peptide Mascot score threshold of ≥20 (corresponds to ~1.4% false discovery rate)

  • Resolve peptide conflicts by assigning shared peptides to the protein with the highest number of unique peptides

• Quantitation and normalization:

  • Sum ion intensities for normalization across samples

  • Generate protein abundance by summing unique peptide ions associated with PBPRA2585

  • Transform data using ArcSinH function for statistical analysis

  • Calculate fold changes between pressure conditions and determine statistical significance using one-way ANOVA

• Validation approaches:

  • Targeted MRM/PRM assays for PBPRA2585-specific peptides

  • Western blotting with specific antibodies as orthogonal validation

  • Correlation with transcriptomic data (acknowledging potential discrepancies)

This comprehensive MS approach has been successfully applied to other P. profundum proteins and enables reliable quantification of pressure-dependent expression changes .

How can ChIP-seq methodology be optimized to study PBPRA2585 genomic binding sites under high-pressure conditions?

Optimizing ChIP-seq for PBPRA2585 under high-pressure conditions requires specialized methodology:

• Cell collection and crosslinking:

  • Design or obtain specialized equipment that allows formaldehyde crosslinking while maintaining pressure

  • Establish optimal crosslinking times specifically for P. profundum cells under pressure

  • Perform rapid decompression only after crosslinking is complete

  • Include controls to assess crosslinking efficiency under different pressure conditions

• Chromatin preparation and immunoprecipitation:

  • Optimize sonication conditions specifically for P. profundum chromatin

  • Validate antibody specificity for PBPRA2585 under native and crosslinked conditions

  • Consider epitope tagging approaches (His, FLAG, etc.) if specific antibodies are unavailable

  • Include spike-in controls from non-piezophilic bacteria for normalization

• Sequencing and data analysis considerations:

  • Compare binding profiles between pressure conditions (0.1 MPa vs. 28 MPa)

  • Correlate binding sites with pressure-responsive genes identified by RNA-seq

  • Perform motif discovery to identify potential pressure-specific binding sequences

  • Integrate with RNA polymerase occupancy data to identify active regulatory regions

• Validation experiments:

  • Perform ChIP-qPCR on selected targets to validate ChIP-seq findings

  • Use reporter assays to confirm functional significance of binding sites

  • Conduct EMSAs with identified binding sequences under different pressures

  • Perform sequential ChIP to identify co-occupancy with other regulators

This approach would provide valuable insights into how PBPRA2585 contributes to P. profundum's remarkable ability to adapt to different pressure environments through genomic regulation .

What experimental design considerations are critical for resolving contradictions in pressure-responsive behavior of PBPRA2585?

Resolving contradictions in PBPRA2585 research requires rigorous experimental design:

• Comprehensive pressure range analysis:

  • Include multiple pressure points (0.1, 5, 10, 15, 20, 28 MPa) rather than just atmospheric and deep-sea conditions

  • Measure both acute responses (minutes to hours) and adaptation responses (days)

  • Monitor reversibility by alternating between pressure conditions

  • Calculate pressure coefficients for various PBPRA2585 activities

• Standardization of experimental variables:

  • Use consistent growth media composition across all studies

  • Harvest cells at standardized growth phases (early/mid/late logarithmic phase)

  • Control temperature precisely during pressure experiments

  • Implement standardized protein purification protocols

• Strain considerations:

  • Compare results between different P. profundum strains (SS9, DSJ4, etc.)

  • Create isogenic mutant strains differing only in PBPRA2585 sequence or expression

  • Include complementation strains to confirm phenotype specificity

  • Consider heterologous expression in non-piezophilic model organisms

• Statistical design and analysis:

• Addressing transcriptomic-proteomic discrepancies:

  • Design time-course experiments to capture temporal dynamics

  • Measure both mRNA and protein levels in the same samples

  • Assess protein post-translational modifications and turnover rates

  • Consider protein localization studies under different pressure conditions

This systematic approach addresses the observation that transcriptomic and proteomic data can show anti-correlation for pressure-responsive proteins in P. profundum , helping to resolve contradictory findings.

How should researchers design experiments to assess the role of PBPRA2585 in pressure adaptation of Photobacterium profundum?

A comprehensive experimental design to assess PBPRA2585's role in pressure adaptation should include:

• Genetic manipulation strategy:

  • Generate PBPRA2585 knockout mutants using allelic exchange

  • Create complemented strains with wild-type and mutated versions

  • Develop conditional expression systems for tight regulation

  • Construct reporter fusions to monitor PBPRA2585 expression in real-time

• Phenotypic characterization under pressure:

  • Growth curve analysis at different pressures (0.1-28 MPa)

  • Microscopic examination of cell morphology and nucleoid structure

  • Stress resistance profiling (temperature, osmotic, oxidative stress)

  • Biofilm formation and motility assays under various pressures

• Molecular analysis framework:

• Integrative data analysis:

  • Identify genes directly and indirectly regulated by PBPRA2585

  • Map PBPRA2585 binding sites relative to pressure-responsive genes

  • Construct regulatory networks through integration of multiple data types

  • Develop predictive models of pressure adaptation involving PBPRA2585

This comprehensive experimental design follows proper experimental design principles and builds on previous studies of P. profundum pressure adaptation , allowing for a thorough assessment of PBPRA2585's specific contributions to this process.

What control proteins and experimental controls should be included when studying PBPRA2585 function?

A robust study of PBPRA2585 requires multiple levels of experimental controls:

• Protein controls for expression and interaction studies:

  • Other known nucleoid-associated proteins from P. profundum

  • Homologous proteins from non-piezophilic bacteria (E. coli, V. cholerae)

  • Housekeeping proteins expected to remain stable under pressure changes

  • Known pressure-responsive proteins as positive controls:

    • DnaK (PBPRA0697) - up-regulated at 28 MPa

    • GroEL (PBPRA3387) - up-regulated at 28 MPa

    • DnaJ - down-regulated at 28 MPa

• Genetic controls:

  • Empty vector controls for complementation studies

  • Point mutations in DNA-binding domains vs. non-DNA-binding domains

  • Strain background controls (wild-type parent for all mutant derivatives)

  • Heterologous expression in non-related bacteria to assess pressure-specific functions

• Methodological controls:

  • Non-specific antibody controls for ChIP experiments

  • Input DNA controls for binding site identification

  • Non-specific DNA sequences for binding specificity assays

  • Mock treatments preserving all experimental conditions except the variable of interest

• Pressure adaptation controls:

  • Pressure time-course experiments with matched time points

  • Parallel cultures maintained at constant pressure as references

  • Non-piezophilic bacteria subjected to the same pressure conditions

  • Multiple pressure conditions beyond just atmospheric and deep-sea

What approaches can resolve contradictory data from different experimental techniques when studying PBPRA2585?

Resolving contradictory data about PBPRA2585 requires systematic methodological approaches:

• Identify specific sources of experimental variation:

  • Pressure equilibration times and rates of pressure change

  • Protein extraction and purification methods

  • Growth phase and media composition differences

  • Strain variation and potential spontaneous mutations

  • Antibody specificity and cross-reactivity issues

• Implement standardization protocols:

  • Establish a consensus protocol for cell growth and harvesting

  • Standardize protein extraction and purification methods

  • Define precise pressure conditions and equilibration times

  • Create reference strain sets available to all researchers

  • Develop validated antibodies or epitope tagging approaches

• Integrate multiple experimental approaches:

  • Compare transcriptomic and proteomic data from the same samples

  • Validate in vitro binding results with in vivo ChIP data

  • Correlate structural studies with functional assays

  • Use genetic approaches to confirm biochemical findings

• Apply advanced reconciliation techniques:

  • Perform meta-analysis of all available data sets

  • Use Bayesian statistical frameworks to integrate diverse data types

  • Implement time-course studies to resolve temporal discrepancies

  • Conduct multi-laboratory validation studies:

This systematic approach addresses the known discrepancies between different experimental techniques that have been observed in P. profundum research, such as the anti-correlation between transcriptomic and proteomic data for stress response proteins .

What are the optimal statistical approaches for analyzing PBPRA2585 MS data across pressure conditions?

For robust statistical analysis of PBPRA2585 MS data across pressure conditions:

• Data preprocessing protocol:

  • Filter MS data to include only multi-charged ions (2+, 3+, 4+)

  • Select the five most intense MS/MS spectra per feature

  • Apply appropriate peptide scoring thresholds (Mascot score ≥20)

  • Resolve peptide conflicts by assigning shared peptides to the protein with the highest number of unique peptides

• Normalization and quantification strategy:

  • Sum ion intensities for normalization across samples

  • Generate protein abundance values by summing unique peptide ions associated with PBPRA2585

  • Transform data using ArcSinH function to address near-zero measurements

  • Calculate fold changes between different pressure conditions

• Statistical testing framework:

  • Apply one-way ANOVA on transformed data for comparison across pressure conditions

  • Use significance criteria: detection by two or more peptides, absolute ratio ≥1.5, p<0.05

  • Implement false discovery rate correction for multiple testing

  • Consider non-parametric tests if data violates normality assumptions

• Advanced analysis techniques:

  • Principal component analysis to identify major sources of variation

  • Hierarchical clustering to identify co-regulated proteins

  • Pathway enrichment analysis for functional interpretation

  • Time-series analysis for adaptation studies

This comprehensive statistical approach has been validated for P. profundum proteomics studies and provides a robust framework for analyzing PBPRA2585 expression across different pressure conditions.

How should researchers interpret PBPRA2585 genomic binding patterns in the context of pressure adaptation?

Interpreting PBPRA2585 genomic binding patterns requires sophisticated analytical approaches:

• Binding site identification and characterization:

  • Identify primary binding motifs under different pressure conditions

  • Map binding sites relative to transcription start sites

  • Determine binding strength changes as a function of pressure

  • Assess binding site conservation across related piezophilic bacteria

• Integration with gene expression data:

  • Correlate binding patterns with pressure-responsive gene expression

  • Classify binding sites as activating, repressing, or neutral based on expression outcomes

  • Identify pressure-specific regulatory modules

  • Construct comprehensive regulatory networks

• Functional analysis framework:

• Evolutionary and comparative analysis:

  • Compare binding patterns with homologous proteins in non-piezophilic bacteria

  • Assess evolutionary conservation of binding sites in pressure-adapted species

  • Identify convergent regulatory solutions to pressure adaptation

  • Model the evolutionary trajectory of pressure-responsive regulation

This integrated approach to interpreting genomic binding patterns will provide insights into how PBPRA2585 contributes to P. profundum's ability to thrive under varying pressure conditions through targeted gene regulation , addressing the fundamental physical differences between marine environments that influence bacterial adaptation.

What are the most promising research directions for understanding PBPRA2585's role in pressure adaptation?

The study of PBPRA2585 in Photobacterium profundum presents several promising research directions:

• Systems biology approaches integrating multiple data types to build comprehensive models of pressure-responsive gene regulation networks involving PBPRA2585.

• Evolutionary studies comparing PBPRA2585 sequence, structure, and function across piezophilic and non-piezophilic bacteria to identify adaptive changes.

• Structure-function analysis under pressure to determine how protein conformation and DNA-binding properties are altered by pressure conditions.

• In situ studies using advanced microscopy to visualize nucleoid organization and PBPRA2585 localization under pressure in living cells.

• Applied research exploring potential biotechnological applications of pressure-adapted DNA-binding proteins in protein engineering and synthetic biology.